This proposal is based on the novel observation that the cell cycle machinery in hair cells is activated following antibiotic administration. We hypothesize that this cell cycle reentry is intimately involved in regulating the subsequent cell death of hair cells. The importance of the cell cycle machinery in stress responses and cell death is well established. Our earlier work suggested a role for the cell cycle machinery, in particular the cyclin-dependent kinase (CDK) inhibitor p19lnk4d, in maintaining the non-dividing state of hair cells and thus in regulating the sensitivity to ototoxic stress and subsequent death. Preliminary results presented in this proposal confirm this suggestion. In response to neomycin, activation of the cell cycle is observed. In addition, inhibitors of the cell cycle machinery are shown to block cell death following administration of aminoglycoside antibiotics. To pursue these observations, we propose three specific aims incorporating a combined pharmacological and genetic approach. In Specific Aim 1, the effect of blocking specific cyclin-dependent kinase (CDK) activity on response to ototoxin will be tested. In Specific Aim 2, we will focus on the response of hair cells to ototoxins and attempt to tie this response to the activation of the cell cycle at the molecular level. Finally, in Specific Aim 3 we propose experiments to test the hypothesis that upon reentry into the cell cycle, hair cells activate the 'downstream' DNA damage checkpoint and that this is the, proximal inducer of the cell death machinery... Loss of sensory hair cells is the leading cause of deafness in humans. The mature mammalian cochlea cannot regenerate its complement of sensory hair cells and thus at present, the only treatment for deafness due to sensory hair cell loss is the use of prosthetics such as hearing aids and cochlear implants. Strategies to protect hair cells from ototoxin induced cell death would allow more aggressive use of chemotherapy agents and antibiotic treatment of infections that are now limited'by -their known ototoxic side effects.